Soybean Allergens and Hypoallergenic Soybean Products ...

J Nutr Sci Vitaminol, 46, 2 71-279, 2000

Review

Soybean Allergens and Hypoallergenic Soybean Products

Tadashi OGAWA,1 Masahiko SAMOT02 and Koji TAKAHASHI3

1Research Institute for Food Science, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan2Protein Development R&D Division, Fuji Oil Co., Ltd., Sumiyoshi-cho, Izumisano, Osaka 598-8540, Japan

3National Agriculture Research Center, Kannon-dai, Tsukuba, Ibaraki 305-0856, Japan

(Received August 26, 2000)

Summary About 15 soybean proteins were shown to be recognized by sera of soybean

sensitive patients with atopic dermatitis. Three of them were identified as major allergens

and designated as Gly m Bd 60K, Gly m Bd 30K, and Gly m Bd 28K, respectively. Gly m Bd

60K is an a subunit of ƒÀ-conglycinin well known as a major soybean storage protein. Gly m

Bd 30K is also known as a soybean oil-body-associated glycoprotein with a molecular

weight of 34,000, which is homologous to Der p (or f) 1, a major allergen of house dust

mite, classified under the papain super family. Gly m Bd 28K is a vicilin-like glycoprotein

with a molecular weight of 26,000, a minor component fractionated into 75 globulin frac

tion. The reduction of allergenicity of soybean and soybean products has been developed

with respect to the above-mentioned major three allergens as the targets by the use of the

combined techniques of a chemical breeding, a physico-chemical treatment, and an enzy

matic digestion. Among the three major allergens, the a subunit of ƒÀ-conglycinin and Gly

m Bd 28K were eliminated from soybean seeds by the development of a mutant line, Tohoku

124, introduced by a chemical breeding technique. The strongest allergen, Gly m Bd 30K,

was almost completely removed from defatted soymilk prepared from Tohoku 124 by a salt

ing-out technique and a centrifugation under the limited pH and ionic strength and alter

natively by an enzymatic digestion. By the application of these procedures, several hypoal

lergenic soybean products, such as cooked soybean grains, soybean curd (Tofu), and fer

mented soybean paste (Miso), soymilk, and a jelly-like soybean cake have been made to eval

uate their usefulness by a challenge test for soybean-sensitive patients. It has been demon

strated by a preliminary trial that about 80% of the soybean-sensitive patients could ingest

these hypoallegenic products without any adverse reactions.

Key Words soybean, allergen, allergenicity, hypoallergenic, chemical breeding

of many foods that elicit adverse reactions, soybean and soybean products are known as one of the major allergenic foodstuffs, especially for Japanese, while they have been recognized as an important protein source. When 86 patients randomly-selected according to their case history (25% of 361 patients diagnosed to have food allergies with atopic dermatitis) were examined by radioallergosorbent test (RAST) using various allergen discs, the incidence of the positive response to the individual food disk was estimated as follows: egg white

(26.7%), soybean (14.0%), wheat (12.7%), milk (11.6%), and rice (8.1%) (1). In recent years, soybean protein isolate (SPI) prepared from defatted soybean meal has been increasingly applied in the field of food

processing because of its high nutritional quality, processing functionality and economical basis, so that it has been difficult for soybean-sensitive patients to select

allergen-free products from commercially-available

processed foods (see Fig. 2). At the present day, a strict elimination of offending foodstuffs from diets is generally adopted as a conventional and effective treatment for it is the only certain prophylactic method in food allergies. The elimination of nutritionally-fundamental or essential foods for extended periods, however, may lead to malnutrition for young patients. There is, therefore, an urgent demand for food scientists to identify the protein components responsible for the allergic manifestation by the ingestion of foods and to reduce the aller

genicity for the healthy use of soybean products by soybean-allergic patients.

In 1934, Duke (2) pointed out soybean as a possible important source of a food allergy among people taking soymilk formula as a milk substitute. In 1980, a soybean allergen had first been demonstrated by Moroz and Yang (3) using a serum of a worker in laboratory, who might have been sensitized through air ways, and the allergen was isolated and identified as Kunitz type soybean trypsin inhibitor (KSTI). Shibasaki et al. (4) also reported that various allergenic protein components occurred in soybean protein fractions and the IgE

Abbreviations: RAST; radioallergosorbent assay, SDS-PAGE;

sodium dodecyl-sulfate polyacrylamide gel electrophoresis,

ECL; enzyme chemiluminescence, ELISA; enzyme-linked im

munosorbent assay, SPI; soybean protein isolate, KSTI; Kunitz

soybean trypsin inhibitor.

271

272 OGAWA T et al.

antibodies in sera of soybean allergic patients showed a

cross reactivity among the 2S-, 7S-, and 11S-globulin

fractions by the RAST inhibition analyses. But this

method could not characterize the individual protein

component responsible for the cross reactivity. He

demonstrated that most allergenic fraction was the 2S

globulin fraction, and 75 and 11S- in this order.

Several investigators wrote about the features of soy

bean allergens, but no detailed information has been

presented. The allergenicity of soybeans is known to re

side in the protein fractions, not in soybean oil itself (5),

whereas oxidized soybean oil has been shown to en

hance IgE-binding ability of soybean or other food pro

teins (6). Burks et al. (7) showed that allergenic proteins

in soybean predominated in the 75 or 111S-globulin

fractions rather than in the 2S-globulin fraction as a re

sult of immunochemical analysis using sera of soybean

sensitive patients with atopic derrmatitis. Recently,

Herian et al. (8) reported that the sera of patients sensi

tive to both peanuts and soybeans bound to several pro

tein components with molecular weights ranging from

50,000 to 60,000 (probably subunits of ƒÀ-conglycinin)

and also to a component with a molecular weight of

about 20,000, not identical with KSTI, which was

strongly recognized by the IgE from the patients allergic

only to soybeans. Rodrigo et al. (9) reported that the in

halation of soybean dust accidentally caused asthma in

Barcelona. The patients with asthma raised against soy

bean dust had specific IgE antibodies for the glycopro

teins with molecular weights lower than 14,000, which

were assumed to be degradation products of ƒÀ-cong

lycinin or unique protein species occurring in soybean

hulls, designated as Gly m 1 and 2. Herian et al. (8) de

scribed different soybean-allergic subjects being sensi

tive to quite different proteins and able to be classified

into three categories according to the immunoblotting

patterns. Our results also showed that the IgE-binding

proteins varied among the patients but the patients

could not be classified into distinct groups according

to their immunoblotting patterns (1). Recently, we

demonstrated the occurrence of about 15 protein com

ponents binding with IgE antibodies in sera of soybean

sensitive patients, three of them named as Gly m Bd

30K, Gly m Bd 28K, and Gly m Bd 60K were shown to

be major allergenic proteins (1). Based on this informa

tion about allergenic proteins in soybeans as the target

to be removed, many approaches to reduce the aller

genicities of soybeans and soybean products have been

proposed; (a) physico-chemical procedures such as heat

denaturation and precipitation, (b) destruction and

modification of allergenic structures such as an intro

duction of polysaccharide moieties and enzymatic di

gestion, (c) breeding (selection of an allergen-deficient

variety or induction of mutants), (d) genetic engineer

ing, and (e) fabrication of non-allergenic constituents.

Furthermore, there have concurrently been developed

more selective and sensitive methods for an evaluation

of allergenicity of soybean products, which can be appli

cable during the course of processing. The convenient

methods to detect and determine the major allergens by

immunoblotting and enzyme-linked immunosorbent assay (ELISA or sandwich ELISA) have been established using allergen specific monoclonal antibodies. The present paper reviews recent information on the molecular, biochemical and immunological properties of the major allergens from soybeans and the development of hy

poallergenic soybean products.

Major Soybean Allergens1) Gly m Bd 30K

The soybean allergenic protein, Gly m Bd 30K (1) which is most strongly and frequently recognized by the IgE antibodies in sera of soybean-sensitive patients with atopic dermatitis, has been characterized as a soybean seed 34 kDa oil-body-associated protein (10). This protein had been identified by Kalinski et al. (11) from the fractionated soybean oil body membrane, whereas the cDNA was isolated and cloned as a vacuolar storage

protein P34 with close homology to thiol proteases classified under a group of papain super family. The primary structure of Gly m Bd 30K was shown to have about 30% homology or 54% similarity with Der p 1, a house dust mite allergen that is thiol protease found in feces of Dermatophagoides preronyssius (12). As shown in Fig. 1 the mature P34 vacuolar protein consists of 257 amino acid residues which is derived by a removal of a

part of N-terminal 122 amino acid residues from a precursor protein with a molecular weight of about 47,000 during the maturation in a vacuole (11). The

glycosylation site of Gly m Bd 30K was established to be located on Asn170 residue of a mature protein (13), which consists of mannose, N-acetylglucosamine, xylose, and fucose in a molar ratio of 3:2:1:1, respectively, indicating one of typical plant aspargine-N linked high mannose type glycans with xylose and fucose branch. The localization of Gly m Bd 30K (P34) in vacuoles of soybean cotyledons was confirmed by an electron microscopic immunostaining technique (14). In recent years, IgE binding sites (B-cell epitopes) located on Gly m Bd 30K were investigated by using synthetic

peptides and identified to be located on the 3-12, 100-110, 229-238, 299-308, and 331-340 amino acid residues, respectively (15). Interestingly, all the epitope sites recognized by human IgE antibodies were shown to be quite different from those on house dust mite allergen, Der p 1. The epitope of the monoclonal antibody of F5 (IgG), which was raised against Gly m Bd 30K using BALB/c mouse, is identified on the 115-132 amino acid residue (16). Gly m Bd 30K was specifically associated with the proteins in 7S-globulin fraction through the disulfide linkage. This property added an important piece of information to the strategy of development of hypoallergenic SPI. Furthermore, there is no soybean variety lacking Gly m Bd 30K in the stock culture of soybean. The cDNA was cloned and the recombinant allergen without glycan moiety was prepared from E. coli, which was recognized by sera of soybeansensitive patients, suggesting that rely m Bd 30K can be applicable for a diagnostic use as an allergen standard of RAST (17). In addition, the distribution of Gly m Bd

Soybean Allergens 273

Fig. 1. Molecular structure of P34 (Gly m Bd 30k). A, Primary structure of P34 (pre and pro-domains indicated by italic) of P34; B, Processing site, domains and glycosylation sites on the molecule of P34 precursor. (adopted from ref. 11).

30K as the index of a soybean allergenicity in soybean varieties and soybean products can be selectively determined by the use of monoclonal antibodies, F5 and H6

(18) (Fig. 2).2) GlymBd28K

A minor protein component in soybean recognized by soybean-sensitive patients with about 25% incidence, one of major allergens named Gly m Bd 28K, was isolated and purified from 7S-globulin fraction pre

pared from defatted soybean flakes (products from Indiana-Ohio-Michigan, U. S. A.) (19). The purified aller

gen was shown to be a glycoprotein with the molecular mass and isoelectric point of 26 kDa and 6.1, respectively and an Asn-N linked glycan moiety with the same sugar composition as that of Gly m Bd 30K was identified to be located on Asn20 residue of Gly m Bd 28K. The N-terminal amino acid sequence analysis gave a result of FHDDEGGDKKSPKSLFMSDSTRVFK and no homolo

gous proteins (peptides) could be found in a data base of proteins (20). However, the translated complementary DNA sequence completely coincides with a part of the sequence of unknown cDNA clones reported from Glycine max (GenBank accession no. A1416520), which is assumed to encode a vicilin-like protein similar to that reported from peanuts (21). When the soybean varieties lacking this 28 kDa allergen were screened in the

Japanese stock cultures and imported soybean seeds, about 80% of varieties examined were shown to lack

Fig. 2. Immunoblotting analyses of Gly m Bd 30K in soybean products. Proteins in the extracts of soybean

products were separated by SDS-PAGE and then blotted on nitrocellulose membranes. The allergen on the membrane was immunostained by a monoclonal anti

body F5. Lanes: a, isolated Gly m Bd 30K: b, soybean

grain: c, soy milk: d. Tofu (soft type): e, Tofu (hard type); f, Kori-tofu (freeze-dried Tofu); g, Kinako (baked soybean); h, Abra-age (fried Tofu); i, Yuba (soy protein coagulant): j, Miso (soybean paste); k, Syoyu (soy sauce); I, Natto (fermented soybean); m, meat ball; n, beef croquette; o, fried chicken; p, fish sausage; q, hamburger. The products lanes a to 1 are the soybean products and lanes m to q are indicated to contain plant

protein isolates as ingredients. (adopted from ref. 46).

274 OGAWA T et al.

Fig. 3. Comparison of the amino acid sequence of the epitope region on a subunit of ƒÀ-conglycinin with the correspon

ding sites of ƒ¿' subunit and phaseolin of Phaseolus vulgaris. A, ƒ¿ subunit of ƒÀ-conglycinin; B, ƒ¿' subunit of ƒÀ-con

glycinin; C, phaseolin. (adopted from ref. 23).

the allergen, Gly m Bd 28K (Takahashi M. personal

communication). The SPI prepared from defatted soy

bean flakes (IOM) was shown to contain this allergenic

protein and the processed foods with plant proteins as

ingredients (SPI) were also demonstrated to contain Gly

m Bd 28K as well as Gly m Bd 30K (22).

3) Gly m Bd 60K (ƒ¿ subunit of ƒÀ-conglycinin)

The other allergenic protein in the 7S-globulin frac

tion, which was recognized by about 25% of sera from

soybean-sensitive patients with atopic dermatitis, was

identified as an a subunit of ƒÀ-conglycinin (23). The IgE

antibodies recognizing the a subunit showed no cross

reactivity against either ƒ¿' or ƒÀ subunit of ƒÀ-con

glycinin known to be highly homologous to a subunit. ƒ¿

Subunit of ƒÀ-conglycinin is a glycoprotein with the mo

lecular weight of 57,000, and with pI of 4.90 (24). The

amino acid sequence of the precursor deduced from the

cDNA consisted of 543 amino acid residues (25). The

epitope(s) of the IgE antibodies were shown to be lo

cated on the peptide of 232-383 residue from N-termi

nal, which is highly homologous to ƒ¿' subunit and

phaseolin, a storage protein of Phaseolus vulgalis (23)

(Fig. 3).

4) Other allergenic proteins in soybean

Soybean low molecular weight proteins identified as

allergens eliciting Barcelona asthma by Rodrigo et al.

(9) Were identified as Gly m 1.0101(Gly m 1A) and Gly

m 1.0102 (Gly m 1B) which are isoforms with different

molecular weights of 7,500 and 7,000, respectively

(26). Their amino acid sequences are well matched to a

part of the hydrophobic protein first reported by Odani

et al. (27), which is synthesized in the endocarp on the

inner ovary wall and is deposited on the seed surface

during development of soybeans (28). Patients with

Barcelona asthma have specific IgE antibodies for this

unique glycoproteins distributed in soybean hulls. Gly

m 2 is demonstrated as an allergen in hulls also related

to Barcelona asthma with a molecular weight of 8,000

and pI of 6.0, which is homologous with a storage pro

tein from cotyledones of Vigna radiata (cow pea) and

with a disease response protein from Pisum sativum

(green pea) (29). However, the cross-reactivity between

those proteins have not been elucidated. Soybean pro

fun was identified as an allergen (Gly m 3) with a mo

lecular weight of 14,000 and pI of 4.4, which is homol

ogous to Bet v 2, a birch pollen allergen, with a se

quence identity of 73% and 11 other plant profilins

with 69 to 88% identity (30). These three allergens are

recognized as inhalant allergens eliciting asthma in

Barcelona when a cargo boat unloads soybean grains,

because the allergenic proteins are located in a part of

the hulls of the grain surface. Several reports on

glycinin as allergens could be found, and acidic sub

units Ala, Alb, A2, A3 and A4 were identified to be al

lergenic (31). The IgE epitope on the acidic chain of

glycinin Gl is located on 192-306 amino acid residue

(32). KSTI was first identified as a soybean allergen

using sera of patients with asthma working in labora

tory and being sensitized through the air way by deal

ing with a fine powder of KSTI as a reagent (3), and also

causing sensitization of occupationally exposed bakers

(33). We examined sera of the patients and found a few

patients have IgE against KSTI (frequency of sensitiza

tion; about 1.5% in soybean-sensitive patients with

atopic dermatitis (1)).

Development of hypoallergenic soybean products

1) Chemical breeding

A new soybean line (GIycine max Tohoku 124) lack

ing the ƒ¿ subunit of ƒÀ-conglycinin was induced by irra

diation with 20 kR (1.0kR/h) gamma-ray to Karikei

434 with a marked decrease in the level of the ƒ¿-, ƒ¿'

and ƒÀ-subunits of ƒÀ-conglycinin (34). The SDS-PAGE

pattern of protein fraction of Tohoku 124 indicates that

the seeds lack one of the major allergens, the a subunit

of ƒÀ-conglycinin (Fig. 4). Recently, it was confirmed that

this mutant Tohoku 124 also lacks another major aller

gen, Gly m Bd 28K, together with the a subunit of ƒÀ

- conglycinin from a result of the immunoblotting analy

sis using monoclonal antibody C5 (19) specific to Gly m

Bd 28K as shown in Fig. 3 (35). This fact indicates that

an application of Tohoku 124 for processing of soybean

products is beneficial for developing hypoallergenic soy

bean products because of the absence of the two major

Soybean Allergens 275

Fig. 4. SDS-PAGE patterns of soybean proteins from various soybean varieties and Tohoku 124. Lanes 1. Suzuyutaka; 2, Tachiyutaka; 3. Kari-kei 434; 4. Tohoku 124. (adopted from ref. 34).

allergens. Gly m Bd 28K and 60K, in advance. However,

a mutant lacking Gly m Bd 30K could hardly be found

even by screening the soybean varieties and mutants

available in the stock culture of the soybean breeding

laboratory ofTohoku National Agricultural Experiment

Station (Takahashi M, personal communication).

2) Physicochemical approach

Heat treatment is a general method in food process

ing and induces denaturation of protein structures.

Epitope structures of most allergens are, however, as

sumed to be sequential, so that the reduction of aller

genicity due to heat denaturation would not be ex

pected. It has been reported that the IgE-binding activ

ity of Gly m Bd 30K is remarkably enhanced by an auto

clave treatment (36). As a unique technique of the hy

poallergenic process, the selective removal of Gly m Bd

30K from soymilk or defatted soymilk by centrifugation

under a specified condition had been achieved. The se

lective removal of Gly m Bd 30K was dependent on the

unique characteristic of solubility different from those

of the major storage proteins, glycinin and ƒÀ-cong

lycinin. In the case of non-defatted soymilk, about 90%

of Gly m Bd 30K could be removed into the oil pad layer

formed by centrifugation in the presence of reducing

agents (37). In the case of defatted soymilk, about 97%

of Gly m Bd 30K could be removed as the precipitate in

the presence of a reducing agent (10 mM sodium bisul

fite) under the specified condition (1 M Na2SO4 in acidic

pH of 4.5). The major storage soybean proteins, both

glycinin and ƒÀ-conglycinin, remained in the super

natant after centrifugation (38). A small amount of Gly

m Bd 30K, however, could not be removed from super

natant in the absence of reducing reagents. It indicates

a possible formation of disulfide linkage between Gly m

Bd 30K and a (ƒ¿') subunits of ƒÀ-conglycinin (39). This

hypothesis was proved by the following fact. By using a

mutant soybean Tohoku 124 lacking the a and ƒ¿' sub

unit, the removal ratio of Gly m Bd 30K from defatted

soymilk was improved to 99.8% from 97% (normal soy

bean) without addition of reducing agents for reductive

cleavage of disulfide linkage between Gly m Bd 30K and

the a or ƒ¿' subunit of ƒÀ-conglycinin (39). Accordingly,

as a result of combination of an application of Toholcu

1.24 and a physicochemical procedure, the substan

tially complete removal of the three major allergenic

proteins (Gly m Bd 30K, a subunit of ƒÀ-conglycinin,

and Gly m Bd 28K) from defatted soymilk was attained

(38) (Fig. 5). The average removal rate of the three al

lergens was attained to almost 99.9% based on the re

sults of the densitometoric measurement of ECL im

munofluorescent intensity on X-ray film (Fig. 5). Since

these procedures for reduction of allergenicity do not

include the methods of modifying protein structures, es

pecially digestive cleavages, the processing functionality

of soybean storage proteins could not be changed to be

applicable for making the traditional soybean products,

for example, Tofu (soybean cake) and Ganmodoki

(cooked soybean cake).

3) Enzymatic digestion

An enzymatic treatment of whole soybean seeds ef

fectively reduces the allergenicity. Autoclaved soybean

was treated by certain proteases from Bacillus sp. at

37•Ž for 20h, which is the same condition as that of

the fermentation procedure for Natto with Bacillus

natto, the Japanese traditional fermented food (40). The

product has a Natto-like texture while it has no Natto

like flavor or taste (plane). When the residual allergens

were examined by the immunoblot and ELISA (ELISA

inhibition), the product showed no binding activity

against monoclonal antibody F5 and patients' sera and

all the proteins in the enzyme-treated soybean grains

were hydrolyzed into the peptides with molecular

weights of less than 10,000 (Fig. 6) Miso (fermented

soybean paste) also showed no residual immuno-reac

276 OGAWA T et al.

Fig. 5. SDS-PAGE and immunoblotting patterns of defatted soy milk. Defatted soy mills with 1 M Na2SO4 without reducing agents was centrifuged to precipitate Gly m Bd 30K. The supernatant was treated with SDS-PAGE sample buffer and then run on a 12% gel for CBB staining (A) and for immunostaining with a monoclonal antibody C5 specific to Gly m Bd 28K (B) and for immunostaining with a monoclonal antibody F5 specific to Gly m Bd 30K (C). Lane 1, IOM soybean;

lane 2, Tohoku 124. (adopted from ref. 35).

Fig. 6. Hydrolysis of soybean proteins by Protease N.

Soybean grains soaked in water overnight were auto

claved at 121•Ž for 20 min. Ten milliliter of protease

solution per gram of soybean (dry weight basis) was

added to soybean grains, which was incubated for

20h at 37•Ž with gentle shaking. Lanes 1, control (0

unit); 2,1•~103 units; 3,5•~103 units; 4,25•~103

units; 5, 12.5•~104 units; M, molecular marker pro

teins. (adopted from ref. 40).

tivity against patients' sera after fermentation for 3

months (Fig. 7) (41). These facts indicate that the fer

mented soybean products, such as Natto, Miso and

Syoyu (soy source), are candidates for naturally-occur

ring hypoallergenic soybean products. Obata et al. (37)

reported the reduction of allergenicity of Tofu by enzy

matic digestion. Tofu was sliced into blocks of 2 cm

thickness and wrapped with cheese cloth dipped in a

protease from Aspergillus soyae. After the treatment for

150h at 4•Ž, the reactivity of allergens was almost

completely reduced against monoclonal antibody spe

cific to Gly m Bd 30K. The product gave a soft texture

like cheese but not Tofu, and no bitter taste which gen

erally appears in hydrolyzates. They also managed to

produce the hypoallergenic Tofu-like-textured food by

use of a coagulant (e. g. polysaccharide) and an enzyme

treated hypoallergenic soymilk (37). Recently, a novel

hydrolytic processing of soybean proteins was reported

(42). Under the limited hydrolytic condition, the selec

tive digestion of ƒÀ-conglycinin (but not glycinin) was at

tained. The key point of selective digestion is based on

the different denaturation temperatures between ƒÀ

- conglycinin and glycinin at neutral pH. The digestion of

denatured soybean proteins could proceed more rapidly

than that of native proteins with proteases at 70•Ž.

Among Bacillus proteases used for the treatment,

Proleather FG-F (Amano Pharmaceutical Co.) was

found to be effective for the selective hydrolysis of Gly m

Bd 30K as well as ƒÀ-conglycinin. The product obtained

was proved to lose its reactivity against the monoclonal

antibody specific to these two allergens and sera of soy

bean-sensitive patients. As a result of the treatment

with proteases under optimum condition, the three

major allergens could be digested. The product contain

ing glycinin showed the processing functionality such

as gelation to produce Tofu and emulsification activity

remained intact (42).

4) Chemical modification

An attempt to mask the allergenic site of soybean

proteins using the Maillard-type polysaccharide conju

gation was examined. Acid-precipitated soybean pro

teins (APP) and galactomannan mixed in weight ratio

of 1:5 were dissolved in water at 10%) (W/V) and

freeze-dried. Maillard reaction was then induced at

60•Ž under 79% relative humidity (RH) in a desiccator

for several days. The allergenic potential of soybean

Soybean Allergens 277

Fig. 7. Fate of allergens in barley-koji Miso during fermentation. Proteins in 0.6mg of Miso paste were applied on each lane. A, Changed in the Gly m Bd 30k contents at various stages of fermentation: B, Proteins stained with CBB: C,

Immunoblotting patterns of Gly m Bd 30K with a monoclonal antibody F5. M, molecular marker proteins: G, isolated Gly m Bd 30k. (adopted from ref. 41).

proteins can be reduced by the conjugation of galac

tomannan residues to APP (43).

5) Extrusion cooking

Ohishi et al. (44) reported that antigenicity of soy

bean meal against calves' sera was reduced to 0.1% of

the original activity by an extrusion cooking with

screws containing kneading-disc elements and die-end

temperatures exceeding 66•Ž. SDS-PAGE analysis of the

cooked meal indicated that the reduction of antigenicity

was due to destruction or modification of protein mole

cules.

Evaluation of hypoallergenic soybean products and perspectives

Hypoallergenic soybean products have been developed and subjected to the evaluation of usefulness under the observation of physicians and dieticians. In vitro examination of IgE binding activity was done by the use of immunoblot or ELISA techniques. In the case of in vivo examination of allergenicity, a single blind food challenge test or open challenge test is practical in evaluating the processed foods. However, the products will be served to patients who have a strict elimination diet of soybeans and soybean products during about 3 weeks under the control of physicians. A challenge test will be carried out after all symptoms disappear from the patients by the elimination of causative diet. As a standard case, the first 5 days the patients received the hypoallergenic soybean products. If no symptomatic change is recognized in the patients during this period, they will receive the control (allergen-containing) diets continuously for an additional 5 days. If some adverse reactions appear in the patients due to the presence of allergens, the challenge test will be stopped. By the preliminary challenge trial it was confirmed that at least 80% of the soybean allergic patients could use these hypoallergenic products without any adverse reactions. Some of the products, evaluation of which have been

completed, await a chance of distribution to the soybean-sensitive patients through the physicians. In addition, further information on soybean allergens, sensitization to soybean allergens, persistence and symptoms of soybean allergy, and diagnostic features are available from a data base in Internet Symposium on Food Allergens (45).

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